1. Field of the Invention
The invention generally relates to compositions and methods for binding to assay substrata, including compositions containing and methods involving proteins, peptides, nucleic acids and other compounds of importance in biochemical assays, for binding to substrata in a stable, protective, and robust manner. In particular, the invention provides compositions of lyotropic liquid and/or, preferably, liquid crystalline materials capable of binding to assay substrata, including compositions containing assay-associated compounds, particularly biomacromolecules. The biomolecules of interest include molecular weight standards, disease markers, and capture compounds such as antibodies, antigens, receptors, ligands, lectins, chimeras, complementary nucleic acids, antisense compounds, avidin, etc. The lyotropic materials are capable of binding to assay substrata, such as that of the chips that are employed for Matrix-Assisted lased Desorption Ionization (MALDI) and Surface-Enhanced Laser Desorption Ionization (SELDI) mass spectroscopy analyses, providing a stable, protective environment for the compounds and a robust means for deposition on the chip with resulting improvement in signal strength and reproducibility. They are also capable of binding to substrata used in more traditional types of protein assays, such as Enzyme-Linked ImmunoSorbent Assays (ELISAs), for effective deposition of reagents and markers, as well as for blocking non-specific binding (NSB).
2. Background of the Invention
Assays based on mass spectroscopy techniques, such as Matrix-Assisted Laser Desorption Ionization (MALDI) and Surface-Enhanced Laser Desorption Ionization (SELDI) are gaining importance in a number of analytical applications, including early detection of cancer, infectious diseases, and other pathological conditions. In mass spec as well as in other assay methods, it can be important to have one or more standards present, added (“spiked”) to the sample fluid, in order to provide for calibration of both the charge/mass ratio and the intensity. However, in such applications, the presence of compounds in biological fluids that can degrade proteins, peptides and other standards is in many cases inevitable; such compounds include proteases, lysozyme, trypsin, nucleases, etc. Facilitating the use of simple, relatively inexpensive, and well-studied standards such as peptides and proteins calls for a method to protect the standard molecule from degradative enzymes and other conditions or compounds, and for accomplishing a high degree of substrate binding for signal enhancement.
In addition, SELDI is a mass spec technique that, through the use of sample substrata with specially tailored surface chemistries, can be of tremendous advantage in selecting desired standards and markers as well as increasing their signal:noise ratios, but is currently not used to full advantage. As an example, in the case where two standard molecules are used in order to provide better calibration, the variance in binding between the two (or more) molecules on SELDI chips, the run-to-run variability of the peak positions and intensities, and variability in the SELDI chips themselves, confounds the calibration of peak positions (m/z ratios) and intensities. This is particularly true in cases where imprecision in calibration of m/z ratios leads to improper integration of peaks.
In the art of laser-desorption mass spectrometry a number of substrates have been developed for selective adsorption of targeted molecules of importance in, e.g., biomedical assays. U.S. Pat. No. 6,579,719 for example describes methods for applying charged and hydrophobic-interaction surfaces for selective capture of biomarkers in the context of laser-desorption mass spectrometry.
In solid-phase assays, there is a need for protein-friendly, even biomimetic, materials and methods for hosting capture molecules and other assay-associated proteins, in such a way that the analyte molecules are captured efficiently and can be brought down to the substrate with high affinity. There is a fundamental challenge in this endeavor which has placed limitations on the quantification, specificity, and ease of use of current methods, and this challenge is very pronounced in certain cases, such as the case of receptor-based assays: namely, by definition, solid-phase assays involve solid-liquid interfaces that tend to denature sensitive proteins such as receptors, as well as other membrane-associated proteins. Indeed, it is well known that membrane-associated proteins tend to denature or flocculate over time even in simple aqueous (buffered) solution, and the more mature techniques in the study of these compounds ensure that at least some lipid is retained in the preparations used in analyses. The analysis of ligand-receptor interactions is of central importance in the screening of potential pharmaceutical actives, and yet there remains a major unsolved problem, at the time of this writing, of how to design a material that will preserve the natural functionality and characteristics of receptor proteins and other functional biomacromolecules and, at the same time, exhibit desired binding to useful substrata. Broadly speaking, a solid surface is an excellent means by which to concentrate species which, in solution or suspension, would be so dilute as to be difficult to quantify, yet the same surface can wreak havoc with delicate proteins such as receptors. Even glycolipid receptors, such as bacterial adhesin receptors, have been shown to yield erroneous, non-physiologic binding selectivity results when used in traditional solid-phase assays, due to improper presentation of the saccharide head groups when the lipid is adsorbed to a solid surface. There is a clear need, particularly in the pharmaceutical industry, for materials that can provide a near-physiologic conformation and presentation of membrane proteins and receptors, preferably with access to both binding and active sites, yielding a degree of fidelity obtainable perhaps only with whole cell-based assays but in a simpler and more controlled system.
Regulatory feedback can alter receptor-based physiological responses, which are further contingent on interactions between different hormonal or signaling systems, and so it is important to interpret, e.g., pharmaceutical screening studies in the context of biochemical data reflecting direct receptor effects of drugs, in purified systems free from extraneous components. Furthermore the need for whole, intact receptor molecules hosted in a physiologic milieu is crucial in view of allosteric effects, competitive binding, multisite binding, desensitization, and other effects that quantitatively and even qualitatively modify binding. Allosteric effects, involving the global protein, which drive signal transduction, are in many receptors driven by the lower free energy associated with binding site/ligand interaction after binding-induced conformational changes; thus, in the absence of the entire protein and associated allosteric effects, studies of competitive binding can be qualitatively incorrect. In addition, with certain multisite receptors, it is known that the natural ligand and exogenous agonists/antagonists can bind to different sites, and so an assay based on a partially expressed protein exhibiting only the natural ligand binding site would yield false negatives with exogenous compounds, and the opportunity afforded by the new potential drug might well be missed. Similarly, in receptors such as the 5-HT-2c receptor, where the binding site involves a transmembrane domain, as well as in cases where the site is at the membrane/water interface or (as in the n-acetylcholine receptor) at the interface between two subunits, it would be erroneous to work only with a partially expressed protein representing a putative binding site. Discrimination between agonist and antagonist binding sites will clearly require intact receptor, and even such events as dimerization of the EGF receptor, which has a strong effect on binding affinity, apparently requires intact receptors, as receptor-related molecules such as the secreted binding domain and gp74v-erbB do not give evidence of dimerization. In view of these facts, there is a need to improve drug-screening assays by satisfying the need for a receptor with its allosteric regulatory mechanism intact, and with proper presentation and accessibility of binding site(s).
Liposomes have been used in conjunction with various biochemical assays, but suffer from instabilities, leakage, opsonization-related problems, incompatibilities with many proteins including membrane-associated proteins, and generally, greatly restricted access to the compounds they encapsulate. Concerning protein incompatibilities, even insulin has been shown to induce leakage of DPPC liposomes through bilayer interactions [Xian-rong et al., Acta Pharm. Sinica (2000) 35(12):924]. These limitations can preclude their use as carriers for bioactive and capture molecules, or at least require tethering of these compounds via laborious and/or expensive conjugation procedures. Use of liposomes in diagnostics is largely limited to the use of high-transition temperature lipid bilayers because of their resistance to instability, rancidification, and opsonization, at least in the case of ready-to-use products. Obviously crystalline materials are essentially non-functional as solvents, and thus integral proteins cannot be incorporated. This in turn largely limits the use of liposomes to the encapsulation of compounds inside the aqueous interior of a rigid liposome, leaving the compound inaccessible to the crucial intermolecular interactions that are central to, for example, immunoassays. Furthermore, the spherical shape of liposomes is simply not conducive to intimate substrate contact.
In summary, it would be a boon for researchers, clinical chemists, pharmaceutical scientists and others dealing with bioassays to have available compositions (e.g. carrier particles) whereby assay-associated molecules could be sequestered, protected, and subsequently deposited reliably on selected substrata.
Simple micelles and microemulsion droplets are not well suited as carrier particles for helping to bind macromolecules to substrata, and also poorly suited for providing a protective encapsulation. Both are very labile, not to be viewed as having any sort of permanence, and in the current theory are viewed as very rapidly exchanging material with each other, with any surfaces present, and with the aqueous domains. And if an ionic interaction between surfactant and substrate were sufficiently strong, the likely result would not be micelles or microemulsion droplets adsorbed to the substrate, but rather individual molecules adsorbed (to form a monolayer, or perhaps multilayer).
In addition, one of the commonly held beliefs by those practiced in the art of MALDI has been that the presence of lipids in samples suppresses ionization and therefore is detrimental to MALDI analysis. Further, since SELDI is a form of MALDI, one might have expected that the addition of lipids to samples would have caused the expected ion suppression. This belief obviously has taught away from the use of lipid-based materials in connection with MALDI and SELDI.
The prior art has thus far failed to provide compositions, and methods for their use, whereby standard molecules can be bound to assay substrata, particularly in a manner whereby the standards are protected and stabilized. Similarly, materials for hosting biospecific capture molecules and other assay-associated compounds, and for sequestering analytes from solution, have suffered from a non-physiologic nature, laborious conjugation procedures, sub-optimal substrate binding, limiting instabilities, poor presentation of binding groups, and/or obstructions to key molecular interactions.